Summary Despite the great potential of human embryonic stem cells (hESCs) for advancing new treatments for human disease, there are important unknowns in understanding the stem cell state. Addressing these unknowns could improve the ability to establish pluripotent stem cells: how to maintain them and how to turn them into specific cells. The proposed research strategy will address questions about pluripotency through study of the Wnt pathway effectors, T cell factor and Lymphoid enhancer factors (TCF/LEFs). One member, TCF3, is a transcription factor that in mouse ESCs (mESCs) represses expression of master pluripotency factors Oct4, Sox2, and Nanog (OSN). Importantly, mouse Tcf3 limits pro-pluripotency feedforward expression of these genes during mESC differentiation. While studies with mESC have been useful and show how Tcf3 should be considered the fourth core stem cell regulatory factor, extrapolating the role to hESCs has inbuilt limitations due to key signaling differences between mESCs and hESCs. But TCF3 function in human ESCs is entirely unknown. Our preliminary data show that TCF3 is dynamically expressed in hESCs. By comparison with other core factors, TCF3 protein levels decline rapidly upon differentiation before other changes are seen and may therefore be a critical, first event. TCF3 expression is highly varied in hESC colonies indicating that hESCs may be poised to differentiate. Differentiation is accompanied by rapid upregulation of other TCF/LEFs suggesting switching of Wnt target gene promoter occupancy. The overarching hypothesis is that hESCs express TCF3 which inhibits Wnt signaling, maintains a stem cell chromatin structure and maintains Activin signaling (a key pathway regulating hESC pluripotency), to hold hESCs in a poised state; pluripotent but ready to go. With differentiation signals TCF3 is replaced by activating TCF/LEFs that alter chromatin structure, driving gene expression and cellular differentiation. Specific Aim 1 will test the role of TCF3 in regulating Activin signaling. We will study control of Activin inhibitors, Follistatin and Inhibin E, identified as potential TCF3 target in preliminary studies. Specific Aim 2 will test the role of TCF3 in regulating the chromatin remodeling factor SMARCA2. These studies could define the relevance of TCF3 downregulation for hESC pluripotency and differentiation and place it in the larger hESC regulatory network. Relationships between TCF3 and master regulators (OSN) will also be defined using microarray and ChIP-Seq. Specific Aim 3 will test the role of TCF3 relatives in hESC differentiation. We find hESC differentiation is accompanied by rapid up-regulation of TCF3 relatives, (LEF-1/TCF-1/TCF-4). Overexpression of these factors will be used to test their role in driving hESC differentiation. Using ChIPseq we will test how TCF/LEFs occupy gene promoters to control genes in cell type- specific ways. This research could better characterize pluripotency molecularly and establish how signaling pathways can instigate fate decisions by rapidly altering transcription factor activity. These studies could provide new insights into mechanisms regulating early human development.